Physiological Mechanisms Of Enhanced Nitrate Nutrition On Root Growth In Rice Genotypes With Different Nitrate Responsivity

Rice is one of the most important grain crops in China and nitrogen (N) is considered as an important limited factor affecting production of cereal crops. Thus, the efficiency of nitrogen adsorption and utilization by rice becomes an important topic in agricultural production system. Nitrogen affects all aspects of plant growth, from metabolism to resource allocation, growth, and development. Nitrogen is the only macronutrient that is commonly available to plants in both oxidized and reduced forms, mainly nitrate (NO3-) and ammonium (NH4+). The most abundant source for N acquisition by plant roots is NO3-in natural aerobic soils, due to intensive nitrification of applied organic and fertilizer N. By contrast, NH4+in waterlogged paddy soils due to the anaerobic soil conditions is the main form of available N. Changes in N forms and heterogeneous distribution in the soil induce various adaptive mechanisms in plants, among which the plasticity of root development is of crucial importance. In several upland species, root proliferation in NO3-rich regions leads to an increased ratio of root surface to explored soil volume, which facilitates the uptake of nutrients. Localized NO3-supply affects root architecture predominantly through its effects on lateral root length and numbers, and that primary root growth is largely unresponsive to NO3-.Meanwhile, rice roots have specialized aerenchyma cells to transport oxygen from the leaf to the rhizosphere, which is then used to oxidize NH4+to NO3-in the rhizosphere. This is to say, rice root is actually exposed to partial NO3-nutrition (PNN).In the present study, hydroponic experiments were carried out to study NO3-as partial N-source nutrition (Partial NO3-nutrition) regulating root growth in rice cultivars with different N-use-efficiency (NUE) and NO3--response (Nanguang, high NUE and NO3--response; Elio, low NUE and NO3--response) at the seedling stage. Cultivars that produced higher yield when N was applied were considered as high NUE, and poor yielding varieties as low NUE. Based on higher grain yield in cv. Nanguang than in cv. Elio gained from multiple years’ and sites’ experiments, cvs. Nanguang and Elio were considered as high and low NUE rice cultivars, respectively. However, compared to Nanguang, higher weight of1000seeds was observed in Elio. Interestingly, higher biomass accumulation rate was recorded in Nanguang than in Elio, which resulted in similar biomass accumulation between two rice cultivars under two N treatments at the heading stage and higher biomass accumulation and grain yield in Nanguang than in Elio under two N treatments at the maturity stage. Also, PNN increased differences in biomass and grain yields between two rice cultivars differing in NUEs. This is to say, the rice cultivar with a higher NUE has a more positive response to PNN than that with a low NUE, suggesting that there might be a relationship between PNN and NUE.The main results are shown as in follows:1. Hydroponic experiments were carried out to study effects of two NH4+/NO3-ratio (100/0and75/25) regulating root growth at the seedling stage in four rice cultivars with different NO3--response (Nanguang, high NO3--response; Elio, Shanghai-97and Liaojing, low NO3--response). Compared to supply of sole NH4+, PNN increased root biomass and nitrogen accumulation of Nanguang50%and79%, respectively, and the total length and number of roots, adventitious roots and lateral roots of the plant increased significantly, however, the mean length of adventitious and lateral roots did not vary much. No such significant effects were observed on other three genotypes of rice. It is clearly indicated that enhanced NO3-nutrient stimulates emergence of adventitious and lateral roots only, but does not promote their elongation. From the findings it is inferred that responsivity of rice to NO3-in root development differs with the genotype and is one of the factors that affect nitrogen use efficiency by rice.2. Hydroponic experiments were carried out to study six ratios of NO3-(changing from0to50%) regulating root growth in cvs. Nanguang and Elio at the late seedling stage. The response of root growth to PNN was investigated and N status and auxin concentration were recorded. No change in root and shoot dry weight was observed in cv. Nanguang when7%and14%NO3-supplied in the solution for5weeks as compared to sole NH4+nutrition. Nitrate-induced increased length of adventitious and lateral roots were14%and27%under7%NO3-in the solution along with NH4+. And1.9-fold and1-fold stimulation in the length of adventitious and lateral roots in cv. Nanguang was achieved by25%NO3-in the solution as compared with7%NO3-in the solution. Stimulated length of adventitious and lateral roots in cv. Nanguang by PNN resulted mainly from root initiation rather than root elongation. Increase of root growth by PNN was closely related to the increase of IAA levels in rice plants. Root biomass in cv. Nanguang was similar to that of cv. Elio under25%and35%nitrate presence in the solution at5weeks and was higher than that in cv. Elio under partial NO3-nutrition at10weeks. The presence of nitrate increased root initiation in rice with a high N-use efficiency, which led to faster biomass accumulation at later growth stages and higher IAA level in the topmost leaves and stems indicates that PNN stimulated IAA biosynthesis in the aerial parts in cv. Nanguang.3. By using a split-root system, root development patterns, expression of nitrate (NO3-)-responsive genes in response to NO3-as partial N-source nutrition (PNN) in two rice cultivars with either high (cv. Nanguang) or low (cv. Elio) response to NO3-were investigated. Compared to supply of sole NH4+, the initiation of lateral roots in cv. Nanguang rather than in cv. Elio was enhanced by PNN after7d cultivation. Meanwhile, expression of NO3--responsive genes in cv. Nanguang was significantly increased by PNN. And significant increments in activities of maximum and active nitrate reductase (NRmax and NRact) and glutamine synthetase (GS) were observed in the root of cv. Nanguang under PNN as compared to the sole NH4+treatment. Nonetheless, no difference in activities of NRmax, NRact and GS were recorded in the root of cv. Elio under two N treatments, Significant increments in15N flux in the root of cv. Nanguang were observed in cv. Nanguang under NO3-supplied nutrition as compared to the sole NH4+treatment. It is concluded that high in NO3-responsive may be potently related with higher nitrogen uptake efficiency, due to NO3-stimulating lateral root initiation.4. By using a split-root system, auxin distribution and related auxin genes in roots in response to NO3-as partial N-source nutrition (PNN) in two rice cultivars with either high (cv. Nanguang) or low (cv. Elio) response to NO3-were investigated. PNN enhanced auxin polar transport from shoots to roots compared to when supplied with sole NH4+supply in cv. Nanguang, but not in cv. Elio. IAA concentration in the roots of cv. Nanguang was24%higher under PNN than that in sole NH4+supply, whereas no difference was observed in cv. Elio. Nitrate supply also enhanced auxin reflux from the auxin maximum at root tip of cv. Nanguang to the lateral root zone. Stronger expression for more auxin influx and efflux transporter genes was recorded under PNN in cv. Nanguang other than in cv. Elio. These results indicate that higher extents of NO3--response was associated with swifter NO3--responsive auxin transport and distribution and higher density of lateral root in cv. Nanguang.5. To enhance our understanding of cytokinin (CTK) regulation and localized supply of different nitrogen forms on the growth of rice seedlings, leaf morphogenesis, expression of adenosine phosphate isopentenyltransferase (IPT), which catalyzes the rate-limiting step of CTK biosynthesis-responsive genes, five cytokinin fractions in xylem sap, root and1st fully expanded leaf in response to NO3-as partial N-source nutrition (PNN) in two rice cultivars were investigated. First fully expanded leaf area in cv. Nanguang rather than in cv. Elio was enhanced by PNN after14d cultivation. Microscopic analysis of the epidermis of1st fully expanded leaf revealed a15%increment of cell number and a12%increment of cell size under PNN as compared to the sole NH4+treatment. Additionally, PNN-induced increased total concentration of CTK fractions in root and1st fully expanded leaf in cv. Nanguang were34%and25%, respectively. Nonetheless, no difference in total concentration of CTK fractions was recorded in cv. Elio under two N treatments. PNN enhanced expression of more CKs biosynthesis-responsive genes was recorded under PNN in cv. Nanguang other than in cv. Elio. These results indicate that PNN treatments resulted in optimal growth of rice with high in NO3-responsivity seedlings might be stimulated CTK biosynthesis in the the root tips and translocated to the shoot meristematic cells via xylem vessels. Since CTK are involved in the regulation of both cell division and cell elongation, it seems likely that the presence of NO3-is required to maintain biosynthesis and root to shoot transfer of CTK at a level that is sufficient to mediate normal leaf morphogensis.In summary, the results showed above indicated that the initiation of lateral roots in cv. Nanguang rather than in cv. Elio was enhanced by PNN after7d cultivation, the initiation of adventitious and lateral roots was enhanced by PNN after5weeks, root biomass in cv. Nanguang was higher than that in cv. Elio under partial NO3-nutrition at10weeks. The presence of nitrate increased root initiation in rice with a high N-use efficiency, which led to faster biomass accumulation at later growth stages. The contrasting effect of PNN on the auxin distribution was further investigated using transgenic plants expressing the β-glucuronidase (GUS) reporter gene under control of DR5promoter which showed that NO3--supplied nutrition enhanced auxin polar transport from the shoot to root and auxin redistribution in rice cultivar with high response to NO3-nutrition. Additionally, PNN might be stimulated CTK biosynthesis in the the roots and translocated to the shoot.